Methods and systems for cell-free nucleic acid processing

Abstract

Methods and systems for targeted detection of circulating tumor DNA (ctDNA) molecules are disclosed herein. A cell-free nucleic acid may be subjected to conditions to increase its methylation level, thereby yielding a hypermethylated cell-free nucleic acid, and subsequently identifying a sequence of the hypermethylated cell-free nucleic acid.

1 - 43 . (canceled) 
     
     
         44 . A method for generating a library of methylated regions, comprising:
 a) subjecting a first population of nucleic acid molecules or derivatives thereof to conditions sufficient to increase methylation levels of at least a portion of said first population of nucleic acid molecules or derivatives thereof, thereby yielding a second population of nucleic acid molecules comprising hypermethylated nucleic acid molecules;   b) incubating at least a portion of said second population of nucleic acid molecules to enrich for methylated nucleic acid molecules of said second population of nucleic acid molecules; and   c) processing said methylated nucleic acid molecules of said second population of nucleic acid molecules to generate a library of methylated regions.   
     
     
         45 . The method of  claim 44 , wherein said nucleic acid molecules or derivatives thereof are cell-free nucleic acid molecules. 
     
     
         46 . The method of  claim 44 , wherein said nucleic acid molecules or derivatives thereof are genomic nucleic acid molecules. 
     
     
         47 . The method of  claim 44 , wherein said first population of nucleic acid molecules or derivatives thereof comprises a first subpopulation of nucleic acid molecules or derivatives thereof and a second subpopulation of nucleic acid molecules or derivatives thereof. 
     
     
         48 . The method of  claim 47 , wherein said first subpopulation of nucleic acid molecules or derivatives thereof comprises cell-free nucleic acid molecules and said second subpopulation of nucleic acid molecules or derivatives thereof comprises genomic nucleic acid molecules. 
     
     
         49 . The method of  claim 44 , wherein said processing of (c) comprises subjecting said methylated nucleic acid molecules of said second population of nucleic acid molecules to conditions sufficient to amplify said methylated nucleic acid molecules of said second population of nucleic acid molecules, thereby generating amplified methylated nucleic acid molecules. 
     
     
         50 . The method of  claim 49 , further comprising sequencing said amplified methylated nucleic acid molecules to generate a plurality of sequencing reads. 
     
     
         51 . The method of  claim 50 , further comprising obtaining a portion of said second population of nucleic acid molecules, thereby generating control nucleic acid molecules. 
     
     
         52 . The method of  claim 51 , further comprising subjecting said control nucleic acid molecules to amplification and sequencing under sufficient conditions to generate a plurality of control sequencing reads. 
     
     
         53 . The method of  claim 52 , further comprising comparing said plurality of sequencing reads to said plurality of control sequencing reads to confirm enrichment of methylated nucleic acid molecules of said second population of nucleic acid molecules. 
     
     
         54 . The method of  claim 44 , further comprising providing (i) sequencing reads from one or more cell-free nucleic acid samples of one or more subjects and (ii) sequencing reads of one or more cell-free nucleic acid samples of one or more healthy subjects. 
     
     
         55 . The method of  claim 54 , wherein said sequencing reads of said one or more subjects are associated with enriched methylated nucleic acid molecules of said one or more subjects and said sequencing reads of said one or more healthy subjects are associated with enriched methylated nucleic acid molecules of said one or more healthy subjects. 
     
     
         56 . The method of  claim 54 , further comprising mapping said sequencing reads of said one or more subjects to said library of methylated regions, thereby generating a first methylation pattern of methylated nucleic acid molecules associated with said one or more subjects. 
     
     
         57 . The method of  claim 56 , further comprising mapping said sequencing reads of said one or more healthy subjects to said library of regions, thereby generating a second methylation pattern of methylated nucleic acid molecules associated with said one or more healthy subjects. 
     
     
         58 . The method of  claim 57 , wherein said first methylation pattern and said second methylation pattern comprise unmethylated states or methylated states. 
     
     
         59 . The method of  claim 57 , further comprising comparing said first methylation pattern to said second methylation pattern to generate one or more differentially methylated regions (DMRs). 
     
     
         60 . The method of  claim 59 , wherein said one or more DMRs are used to classify a diseased sample and a healthy sample. 
     
     
         61 . The method of  claim 54 , wherein said one or more subjects have or are suspected of having a disease or condition. 
     
     
         62 . The method of  claim 61 , wherein said disease or said condition is a cancer or a tumor. 
     
     
         63 . The method of  claim 50 , wherein said sequencing comprises narrow peak calling or broad peaking calling to identify methylation signals of said plurality of sequencing reads.